The ARD peptide HBc147-183 was shown to be capable of binding to LPS and Lipid A in several different in vitro binding assays.

For each assay, samples were always measured in triplicates. (A) The cartoon illustrates the in vitro assays of peptide-LPS and peptide-Lipid A binding as well as LPS/Lipid A competition. (B) Constant amount of LPS was incubated with increasing concentrations of biotinylated ARD HBc 147-183 peptide on the streptavidine-conjugated beads (0, 0.004, 0.02, 0.1, 0.5 and 2.5 µM). Unbound LPS in the supernatant was measured with the LAL ELISA assay (Materials and Methods). The EU values were normalized with a control without peptide treatment. HBc147-183 8p (containing 8 phosphorylated amino acids) was also included as a control peptide due to its poor binding with LPS. (C) Beads-bound LPS was released into the supernatant by overnight digestion with trypsin agarose. Free LPS in the supernatant was analyzed with the LAL ELISA assay. Released LPS in the supernatant appeared to be in proportion to the amount of ARD peptide HBc147-183 on the beads. (D) Constant amount of Lipid A was incubated with increasing concentrations of HBc147-183 and HBc147-183 8P, respectively. The supernatant was also detected with LAL ELISA reagent. The result here is consistent with the notion that Lipid A can bind to HBc147-183 directly. (E) LPS/Lipid A competition assay. Constant amount of LPS (1 µg) was coated on each well on the ELISA plate, and then incubated with a reaction mixture containing constant amount of 10 nM HBc147-183 and increasing concentrations of Lipid A. The gradual increase of Lipid A reduced the amount of plate-bound ARD peptide HBc147-183 in a dose dependent manner.